The finite nature of fossil fuels, as well as rising prices and environmental concerns, has spurred research to develop chemical production alternatives that are more sustainable. One such alternative is to use engineered microorganisms to convert renewable growth substrates (e.g. sugars) to metabolic products of interest. Using modern genetic techniques and synthetic biology approaches, microorganisms have been engineered to produce a wide variety of chemicals from renewable starting materials (Keasling 2012; Dellomonaco et al., 2010). Metabolic engineering offers the ability to tailor the flow of carbon to desired compounds and leverage the advantages of enzymatic biocatalysts (e.g. specificity, precision, complexity). If economic and productivity targets can be met, engineered microbes could play a large role in replacing the fraction of petroleum used to produce the chemical building blocks that enable current lifestyles.
In recent years, significant effort has focused on producing hydrophobic compounds via fatty acid biosynthesis for use as liquid transportation fuels or commodity chemicals (Lennen and Pfleger 2013). Aliphatic compounds such as fatty alcohols also have applications as detergents, emulsifiers, lubricants, and cosmetics. While fatty alcohols normally make up about 3-5 percent of the final formulation of these products, some such as solid anti-perspirants contain up to 25% fatty alcohols (Mudge et al., 2008). As of 2006, over 1.3 million tons of fatty alcohols were used worldwide each year (Mudge et al., 2008). As a whole, the industry represents over a 3 billion dollar market (Rupilius and Ahmad, 2006). Currently, fatty alcohols are produced either through processing natural fats and oils (oleochemicals) or from petrochemicals (e.g. crude oil, natural gas). In the oleochemical route, fatty acids or fatty acid methyl esters are released from triglycerides and hydrogenated to form fatty alcohols (Matheson 1996). In one common petrochemical route, paraffins are separated from kerosene, then converted to olefins, before being converted to fatty alcohols. As both processes require either modifications to biodiesel or petrochemical fuel stocks, microbial production of fatty alcohols from renewable sugars is a promising alternative.
Fatty alcohols can be generated by microorganisms endogenously (FIG. 1A) via reduction of fatty aldehydes that are made via reduction of acyl-thioesters (coenzyme A or acyl-carrier protein) (Reiser and Somerville 1997). Alternatively, fatty acids have been shown to be directly converted to fatty aldehydes via the action of a carboxylic acid reductase (Akhtar et al., 2013). Genes encoding long chain acyl-CoA reductase activity have been isolated from many organisms including bacteria (Reiser and Somerville 1997), insects (Liénard et al., 2010), birds (Hellenbrand et al., 2011), mammals (Cheng and Russell 2004), and protists (Teerawanichpan and Qiu, 2010). Many of these enzymes are used to synthesize fatty alcohols as precursors to wax esters. Three exemplary classes of reductases include reductases from soil bacteria (Reiser and Somerville, 1997; Steen et al., 2010), reductases from plants such as Arabidopsis or Simmondsia (Doan et al., 2009; Rowland and Domergue, 2012), and reductases found in marine bacteria (Willis et al., 2011; Hofvander et al., 2011). These classes differ in their ability to catalyze multiple reactions and in their substrate preference. Reductases similar to those found in Acinetobacter contain only the domain to catalyze conversion of acyl-thioesters to fatty aldehydes. Conversely, reductases from plants can catalyze both reductions, but generally do not have broad substrate specificity, preferring the dominant long acyl chains found in lipids. Reductases from marine bacteria catalyze both reductions and are active on a wide range of chain lengths.
While fatty acids have been produced with yields of greater than 0.2 g fatty acid per gram carbon source consumed (Dellomonaco et al., 2011; Zhang et al., 2012), the highest reported yields of fatty alcohols have been at least five fold lower. The work of Steen et al. (Steen et al., 2010) demonstrated that fatty alcohols can be produced with titers of around 60 mg/L fatty alcohol and yields of less than 0.005 g fatty alcohol/g carbon source. Further metabolic engineering and fermentation efforts have increased the titer to ˜450 mg/L, but with no significant improvement in yield (Zheng et al., 2012). Alternative strategies have led to slightly higher fatty alcohol yields from a defined carbon source. One strategy reached ˜350 mg/L with a yield of 0.04 g fatty alcohol/g carbon source (Akhtar et al., 2013). Another strategy achieved between 0.04 and 0.055 g fatty alcohol/g carbon source consumed (Dellomonaco et al., 2011). However, greater titers and yields are required if microorganism-based production of fatty alcohols is to replace fossil fuel-based production.